Sporulation in Bacillus subtilis has been studied extensively as a model for cellular development. Initiation of sporulation depends on sensor histidine-kinases (His-kinases), which autophosphorylate, initiating phosphate transfer through an expanded two-component signaling system, the phosphorelay. Subsequent stages of sporulation are controlled by a cascade of sporulation-specific RNA polymerase ??factors, which is initiated by ?F. The activity of ?F is regulated in a compartment-specific manner by a complex network of accessory factors, including the SpollE phosphatase. Here, we propose biochemical and structural approaches to address the structure/function relationship for two key regulators of Bacillus sporulation, the sensor His-kinase KinB, and SpollE, using the thermophile B. stearothermophilus (Bst) as a model system for B. subtilis and the category A pathogen B. anthracis. Specifically, we propose to: 1. Determine the crystal structure of the Bst Sda/KinB complex. Sda is a 46-residue polypeptide that inhibits KinB in response to DNA damage. We have obtained crystals of the soluble, cytoplasmic catalytic core of KinB bound to Sda that diffract beyond 2 A-resolution. 2. Determine the crystal structure of KinB trapped in the act of autophosphorylation. The catalytic core of the His-kinases comprises two domains, the His-phosphotransferase domain (Hpt; harboring the phospho-acceptor His residue) and the ATP-binding kinase domain. A large scale conformational change is required to reposition the kinase domain with respect to the Hpt domain for autophosphorylation to occur. We propose two strategies to trap this transient conformational state for structural studies.; 3. Determine crystal structures of Sda/KinB/Spo0F complexes. To understand the structural basis for phosphotransfer from the Hpt to the response regulator Spo0F, we propose to solve the ternary complex structure with unphosphorylated KinB (preliminary crystals have been obtained), as well as with a stable phospho-His analog, (4'-phospho-2'-furyl)alanine, which will be incorporated into the KinB Hpt by expressed protein ligation.; 4. Structurally characterize the ?F regulatory system (?F/SpollAA/SpollAB/SpollE) controlling the initiation of Bacillus sporulation. [unreadable] [unreadable] [unreadable]